AIMTo investigate the prevalence of high levels of sperm DNA damage among men from infertile couples with both normal and abnormal standard semen parameters.

METHODSA total of 350 men from infertile couples were assessed. Standard semen analysis and sperm chromatin structure assay (SCSA) were carried out.

RESULTSNinety-seven men (28% of the whole study group) had a DNA fragmentation index (DFI)> 20%, and 43 men (12%) had a DFI>30%. In the group of men with abnormal semen parameters (n = 224), 35% had a DFI>20%, and 16% had a DFI>30%, whereas these numbers were 15% and 5%, respectively, in the group of men with normal semen parameters (n=126). Men with low sperm motility and abnormal morphology had significantly higher odds ratios (ORs) for having a DFI>20% (4.0 for motility and 1.9 for morphology) and DFI>30% (6.2 for motility and 2.8 for morphology) compared with men with normal sperm motility and morphology.

CONCLUSIONIn almost one-third of unselected men from infertile couples, the DFI exceeded the level of 20% above which, according to previous studies, the in vivo fertility is reduced. A significant proportion of men with otherwise normal semen parameters also had high sperm DNA damage levels. Thus, the SCSA test could add to explaining causes of infertility in cases where semen analysis has not shown any deviation from the norm. We also recommend running the SCSA test to choose the appropriate assisted reproductive technique (ART).

Chromatin ; pathology ; DNA Damage ; Female ; Humans ; Infertility, Male ; epidemiology ; genetics ; physiopathology ; Male ; Prevalence ; Semen ; cytology ; Spermatozoa ; physiology

AIMTo study the molecular mechanism of epididymal protease inhibitor (Eppin) modulating the process of prostate specific antigen (PSA) digesting semenogelin (Sg).

METHODSHuman Sg cDNA (nucleotides 82-849) and Eppin cDNA (nucleotides 70-723) were generated by polymerase chain reaction (PCR) and cloned into pET-100D/TOPO. Recombinant Eppin and Sg (rEppin and rSg) were produced by BL21 (DE3). The association of Eppin with Sg was studied by far-western immunoblot and radioautography. In vitro the digestion of rSg by PSA in the presence or absence of rEppin was studied. The effect of anti-Q20E (N-terminal) and C-terminal of Eppin on Eppin-Sg binding was monitored.

RESULTSEppin binds Sg on the surface of human spermatozoa with the C-terminal of Eppin (amino acids 75-133). rSg was digested with PSA and many low molecular weight fragments were produced. When rEppin is bound to rSg, then digested by PSA, incomplete digestion and a 15-kDa fragment results. Antibody binding to the N-terminal of rEppin did not affect rSg digestion. Addition of antibodies to the C-terminal of rEppin inhibited the modulating effect of rEppin.

CONCLUSIONEppin protects a 15-kDa fragment of rSg from hydrolysis by PSA.

Animals ; Antibodies ; pharmacology ; Autoradiography ; Humans ; Hydrolysis ; Male ; Prostate-Specific Antigen ; metabolism ; Proteinase Inhibitory Proteins, Secretory ; genetics ; immunology ; metabolism ; Rabbits ; Recombinant Proteins ; genetics ; metabolism ; Semen ; cytology ; metabolism ; Seminal Vesicle Secretory Proteins ; metabolism ; Spermatozoa ; metabolism

AIMTo determine whether testosterone is involved in morphine withdrawal syndrome (WS).

METHODSIn order to induce dependency, rats were treated with subcutaneous injection of morphine (days 1-2, 5 mg/kg; days 3-5, 7.5 mg/kg; days 6-8, 10 mg/kg), and after the last dose of morphine (day 8) WS was induced by intraperitoneal injection of naloxone (1 mg/kg). Wet dog shake (WDS), abdomen writhing (AW), and jumps (J) were recorded as indicators of WS.

RESULTSThe severity of WDS, AW, and J in male rats was greater than that in females. Accordingly, in 4-week castrated and flutamide-treated (10 mg/kg/day for 8 days, i.p.) male rats, WDS, AW, and J were significantly decreased compared to male control rats. Testosterone replacement therapy (10 mg/kg/day for 8 days, i.m.) in 4-week castrated rats restored the severity of WDS, AW, and J behaviors to the level of non-castrated male rats, whereas testosterone potentiated the WDS behavior in non-castrated male rats.

CONCLUSIONIt can be concluded that testosterone might be effectively involved in morphine WS.

Androgen Antagonists ; pharmacology ; Androgens ; pharmacology ; physiology ; Animals ; Behavior, Animal ; Female ; Flutamide ; pharmacology ; Male ; Morphine ; pharmacology ; Morphine Dependence ; physiopathology ; Naloxone ; pharmacology ; Narcotic Antagonists ; pharmacology ; Narcotics ; pharmacology ; Orchiectomy ; Rats ; Rats, Wistar ; Severity of Illness Index ; Substance Withdrawal Syndrome ; physiopathology ; Testosterone ; pharmacology ; physiology

AIMTo investigate the expression of cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR) and the possible mechanism in the development in androgen independent prostate cancer (AIPC).

METHODSImmunohistochemistry was performed on paraffin-embedded sections with goat polyclonal against COX-2 and mouse monoclonal antibody against EGFR in 30 AIPC and 18 androgen dependent prostate cancer (ADPC) specimens. The effect of epidermal growth factor (EGF) treatments on the expression of COX-2 and signal pathway in PC-3 and DU-145 cells was studied using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. ELISA was used to measure prostaglandin E2 (PGE2) levels in the media of PC-3 and DU-145 incubated with EGF for 24 h.

RESULTSCOX-2 was positively expressed in AIPC and ADPC, which were predominantly in endochylema of prostate cancer (PCa) cells. Intense staining was seen in AIPC (80%) and in ADPC (55.5%), but there was no significant association between the two groups. EGFR expression was also positive in the two groups (61.8% in ADPC and 90% in AIPC, P < 0.01). A significant association was found between EGFR expression and a higher Gleason score (P < 0.05) or tumor stage (P < 0.05). The expression of PGE2 was increased in PC-3 and DU-145 cells after being incubated with EGF. Both p38MAPK and PI-3K pathway were involved in the PC-3 cell COX-2 upregulation course. In DU-145, only p38MAPK pathway was associated with COX-2 upregulation.

CONCLUSIONEGFR activation induces COX-2 expression through PI-3K and/or p38MAPK pathways. COX-2 and EGFR inhibitors might have a cooperative anti-tumor effect in PCa.

Aged ; Aged, 80 and over ; Androgens ; metabolism ; Cell Line, Tumor ; Culture Media, Serum-Free ; Cyclooxygenase 2 ; genetics ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Phosphatidylinositol 3-Kinases ; metabolism ; Prognosis ; Prostatic Neoplasms ; genetics ; metabolism ; Receptor, Epidermal Growth Factor ; genetics ; metabolism ; Signal Transduction ; physiology ; p38 Mitogen-Activated Protein Kinases ; metabolism

AIMTo investigate whether adriamycin induces DNA damage and the formation of gammaH2AX (the phosphorylated form of histone H2AX) foci in mature spermatozoa.

METHODSHuman spermatozoa were treated with adriamycin at different concentrations. gammaH2AX was analyzed by immunofluorescent staining and flow cytometry and double-strand breaks (DSB) were detected by the comet assay.

RESULTSThe neutral comet assay revealed that the treatment with adriamycin at 2 microg/mL for different times (0.5, 2, 8 and 24 h), or for 8 h at different concentrations (0.4, 2 and 10 microg/mL), induced significant DSB in spermatozoa. Immunofluorent staining and flow cytometry showed that the expression of gH2AX was increased in a dose-dependent and time-dependant manner after the treatment of adriamycin. Adriamycin also induced the concurrent appearance of DNA maintenance/repair proteins RAD50 and 53BP1 with gammaH2AX in spermatozoa. Wortmannin, an inhibitor of the phosphatidylinositol 3-kinase (PI3K) family, abolished the co-appearance of these two proteins with gammaH2AX.

CONCLUSIONHuman mature spermatozoa have the same response to DSB-induced H2AX phosphorylation and subsequent recruitment of DNA maintenance/repair proteins as somatic cells.

Androstadienes ; pharmacology ; Antibiotics, Antineoplastic ; pharmacology ; Cells, Cultured ; Comet Assay ; DNA Breaks, Double-Stranded ; drug effects ; DNA Damage ; DNA Repair Enzymes ; metabolism ; DNA-Binding Proteins ; metabolism ; Doxorubicin ; pharmacology ; Drug Interactions ; Flow Cytometry ; Histones ; metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; metabolism ; Male ; Phosphorylation ; drug effects ; Protein Kinase Inhibitors ; pharmacology ; Spermatozoa ; cytology ; drug effects ; metabolism ; Tumor Suppressor p53-Binding Protein 1

AIMTo investigate the expression of Spindlin 1 (Spin 1) isoform2 and assess its function in mouse testis.

METHODSFirst, reverse-transcription polymerase chain reaction (RT-PCR) was used to determine whether Spin1 isoform2 is present in mouse testis. Then the expression patterns of the isoform between newborn and adult mice testes were compared by immunoblot analysis. Finally, the diversity of its localization in mice testes at different ages (days 0, 7, 14, 21, 28 and 60) was observed by immunohistochemistry. The localization of the protein in mouse sperm was also investigated by immunofluorescence.

RESULTSThe RT-PCR results show that Spin1 isoform2 is present in mouse testis. As shown by immunoblot analysis, the isoform was more highly expressed in adult testes compared with newborn testes. Interestingly, Spin1 isoform2 did not show up in the cytoplasm of primary spermatocytes until day 14. Also, the protein exists at the tail of the mouse sperm.

CONCLUSIONSpin1 isoform2 is a protein expressed highly in adult testis, which might be involved in spermatogenesis and could be necessary for normal sperm motility.

Age Factors ; Animals ; Animals, Newborn ; Blotting, Western ; Cell Cycle Proteins ; genetics ; metabolism ; Cytoplasm ; metabolism ; Female ; Immunohistochemistry ; Male ; Meiosis ; physiology ; Mice ; Mice, Inbred ICR ; Microtubule-Associated Proteins ; genetics ; metabolism ; Phosphoproteins ; genetics ; metabolism ; Pregnancy ; Reverse Transcriptase Polymerase Chain Reaction ; Sperm Motility ; physiology ; Spermatogenesis ; physiology ; Testis ; physiology

AIMTo identify and define prostate and seminal vesicle abnormalities in patients with chronic male accessory gland infection (MAGI) who failed to respond to antibacterial treatment.

METHODSWe selected 67 consecutive patients with MAGI and persistently elevated bacteriospermia (=or>10(6) colony forming units [CFU]/mL) after three antibiotic courses. Fourteen infertile patients with initial chronic microbial (=or>10(6) CFU/mL) MAGI who responded to antibacterial treatment (<10(3) CFU/mL) served as a control group. All patients and controls underwent transrectal ultrasonography (TRUS) scans and semen analysis. Patients with low seminal plasma volume (<1.5 mL) underwent both pre-ejaculatory and post-ejaculatory TRUS examination.

RESULTSTRUS revealed multiple abnormalities indicative of: (i) bilaterally extended prostato-vesiculitis (group A: 52 cases, 77.6%) (nine of these patients also had micro-emphysematous prostate abscess); and (ii) prostato-vesiculitis with unilateral or bilateral sub-obstruction of the ejaculatory ducts (group B: 15 cases, 22.4%). Mean sperm concentration, total sperm number, ejaculate volume and pH value were significantly higher in group A than in group B. In addition, sperm forward motility and the percentage of normal forms were significantly worse than in controls, whereas leukocyte concentration was significantly higher in group A. Group B patients had all sperm parameters, but their pH values, significantly different from those of controls.

CONCLUSIONAlthough antibiotic therapy is considered suitable when microbial MAGI is suspected, it is impossible to account for a poor response to antibiotics merely on the basis of conventional criteria (clinical history, physical and ejaculate signs). Thus, TRUS may be helpful in the follow-up of these patients.

Adult ; Anti-Bacterial Agents ; therapeutic use ; Bacterial Infections ; complications ; drug therapy ; Epididymis ; diagnostic imaging ; microbiology ; Epididymitis ; complications ; diagnostic imaging ; Follow-Up Studies ; Humans ; Infertility, Male ; diagnostic imaging ; microbiology ; Male ; Prostate ; diagnostic imaging ; microbiology ; Prostatitis ; complications ; diagnostic imaging ; Rectum ; diagnostic imaging ; Seminal Vesicles ; diagnostic imaging ; microbiology ; Ultrasonography ; methods

AIMTo analyze factors influencing the efficacy of hormonal suppression of spermatogenesis for male contraception.

METHODSA nested case-control study was conducted, involving 43 subjects, who did not achieve azoospermia or severe oligozoospermia when given monthly injections of 500 mg testosterone undecanoate (TU), defined as partial suppressors compared with 855 subjects who had suppressed spermatogenesis (complete suppressors). Sperm density, serum testosterone, luteinizing hormone (LH) and follicle stimulating hormone (FSH) concentrations at the baseline and the suppression phase were compared between partial and complete suppressors. Polymorphisms of androgen receptor (AR) and three single nucleotide variants and their haplotypes of FSH receptor (FSHR) genes determined by polymerase chain reaction (PCR) and DNA sequencing technique were compared between 29 partial and 34 complete suppressors.

RESULTSBaseline serum LH level was higher and serum LH as well as FSH level during the suppression phase was less suppressed in partial suppressors. Additionally, in a logistic regression analysis larger testis volume, higher serum FSH concentrations alone, or interaction of serum LH, FSH, testosterone and sperm concentrations were associated with degree of suppression. The distribution of polymorphisms of AR or FSH receptor genes did not differ between partial and complete suppressors. In cases with incomplete FSH suppression (FSH 0.2 IU/L), the chances of reaching azoospermia were 1.5 times higher in the subjects with more than 22 CAG triplet repeats.

CONCLUSIONPartial suppression of spermatogenesis induced by 500 mg TU monthly injections is weakly influenced by hormonal and clinical features but not polymorphism in AR and FSHR genes.

Adult ; Azoospermia ; chemically induced ; genetics ; Case-Control Studies ; Contraceptive Agents, Male ; administration & dosage ; Drug Resistance ; genetics ; Follicle Stimulating Hormone ; blood ; Haplotypes ; Humans ; Luteinizing Hormone ; blood ; Male ; Polymorphism, Single Nucleotide ; Predictive Value of Tests ; Promoter Regions, Genetic ; genetics ; Receptors, Androgen ; genetics ; Receptors, FSH ; genetics ; Sperm Count ; Spermatogenesis ; drug effects ; genetics ; Testosterone ; administration & dosage ; analogs & derivatives ; blood ; Trinucleotide Repeats

AIMTo investigate the transformation of characteristics of epidermal cells from foreskin which were used to reconstruct male rabbit anterior urethra in combination with acellular collagen matrices.

METHODSIn three rabbits, autologous foreskin epidermal cells were isolated, expanded in vitro, and seeded (inoculated) onto a tubular acellular collagen matrix, acquired from allogeneic rabbit bladder submucosa. A urethral mucosal defect was created, and urethral reconstruction was performed with the tubular acellular collagen matrix seeded with epidermal cells.

RESULTSOn gross examination at 12 months following the procedure, the mucosa of the urethral grafts appeared lubricous and smooth. Urethrography showed that a wide urethral caliber had been maintained without any sign of strictures. Histological examination showed a transitional cell layer in the graft without evidence of a margin between the graft and the host tissue at 12 months postoperatively.

CONCLUSIONEpidermal cells seeded onto acellular collagen matrices can be successfully used to reconstruct urethras that have defects and are transformed to transitional epithelial cells.

Animals ; Cell Transplantation ; methods ; Collagen ; Epidermis ; cytology ; Foreskin ; cytology ; Graft Survival ; Male ; Mucous Membrane ; cytology ; Rabbits ; Reconstructive Surgical Procedures ; methods ; Tissue Culture Techniques ; Tissue Engineering ; methods ; Urethra ; surgery ; Urethral Stricture ; surgery

AIMTo characterize mouse capping protein alpha3 (CPalpha3) during spermatogenesis and sperm maturation.

METHODSWe produced rat anti-CPalpha3 antiserum and examined the expression of CPalpha3 in various mouse tissues using Western blot analysis and the localization of CPalpha3 in testicular and epididymal sperm using immunohistochemical analyses. We also examined how the localization of CPalpha3 and beta-actin (ACTB) in sperm changed after the acrosomal reaction by performing immunohistochemical analyses using anti-CPalpha3 antiserum and anti-actin antibody.

RESULTSWestern blot analysis using specific antiserum revealed that CPalpha3 was expressed specifically in testes. Interestingly, the molecular weight of CPalpha3 changed during sperm maturation in the epididymis. Furthermore, the subcellular localization of CPalpha3 in sperm changed dynamically from the flagellum to the post-acrosomal region of the head during epididymal maturation. The distribution of ACTB was in the post-acrosomal region of the head and the flagellum. After inducing the acrosomal reaction, the CPalpha3 and ACTB localization was virtually identical to the localization before the acrosomal reaction.

CONCLUSIONCPalpha3 might play an important role in sperm morphogenesis and/or sperm function.

Acrosome Reaction ; physiology ; Actins ; metabolism ; Animals ; Blotting, Western ; CapZ Actin Capping Protein ; metabolism ; Cells, Cultured ; Epididymis ; cytology ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Sperm Head ; metabolism ; Sperm Tail ; metabolism ; Spermatogenesis ; physiology ; Spermatozoa ; cytology ; metabolism ; Testis ; cytology ; metabolism